Dynamic conformational changes of the P-S6 linker of the pacemaker (HCN) channels identified by sulfhydryl modification: pore-to-gate coupling model

Abstract

INTRODUCTION: The hyperpolarization-activated cyclic nucleotidemodulated (HCN) channel gene family encodes the funny current, a key player of cardiac pacing. Although the topology of HCN channels resembles voltage-gated potassium (Kv) channels, much less is known about their structure-function correlation. We have recently reported various lines of biophysical evidence that supports an evolutionary conserved external pore-to-gate mechanism of HCN channels despite their activation by opposite voltages. METHODS: Here we probed the P-S6 region of HCN1 channels homologous to external pore helix of KcsA potassium channel by cysteine scanning mutagenesis and hydrophilic sulfhydrylreactive 2-trimethylammoniumethylmethane thiosulfonate (MTSET). RESULTS: The introduced cysteines at position 353 and 354, which are closest to the GYG motif, were accessible by external MTSET, which led to reductions in whole cell current for 72 % and 41 % respectively. These reductions in whole cell currents were primarily resulted from negative shift in the activation gating, $V½ ~ -24 mV in Q353C and ~ -9 mV in A354C. More importantly, the MTSET reactivity were state-dependent suggestive of dynamic conformational change of this outer pore structure. These data were in accord with the notion that hindered pore movements affect gating. Interestingly, two other cysteine residues (S357C and M358C) closest to the S6 exhibited no observable effects to MTSET. CONCLUSIONS: We conclude that residues of immediate vicinity of the GYG motif in P-S6 loops of HCN1 channels exhibit statedependent external accessibility to MTSET and that the external pore vestibule and the activation gating of HCN channels are allosterically coupled.